scholarly journals Interchangeability of Periplasmic Adaptor Proteins AcrA and AcrE in forming functional efflux pumps with AcrD in Salmonella Typhimurium

2021 ◽  
Author(s):  
Ilyas Alav ◽  
Vassiliy N Bavro ◽  
Jessica M A Blair
Keyword(s):  
Drug Targets ◽  
Efflux Pump ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Efflux Pumps ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Interaction Sites ◽  
Functional Complex ◽  
Rnd Transporter

Background RND efflux pumps are important mediators of antibiotic resistance. RND pumps including the principal multidrug-efflux pump AcrAB-TolC in Salmonella, are tripartite systems, with an inner membrane RND-transporter, a periplasmic adaptor protein (PAP) and an outer membrane factor (OMF). We previously identified the residues required for binding between the PAP AcrA and the RND-transporter AcrB and have demonstrated that PAPs can function with non-cognate transporters. AcrE and AcrD/AcrF are homologues of AcrA and AcrB, respectively. Here, we show that AcrE can interact with AcrD, which does not possess its own PAP, and establish that the residues previously identified in AcrB-binding are also involved in AcrD-binding. Methods The acrD and acrE genes were expressed into a strain lacking acrABDEF (Δ3RND). PAP residues involved in promiscuous interactions were predicted based on previously defined PAP-RND interactions and corresponding mutations generated in acrA and acrE. Antimicrobial susceptibility of the mutant strains was determined. Results Co-expression of acrD and acrE significantly decreased susceptibility of the Δ3RND strain to AcrD substrates showing that AcrE can form a functional complex with AcrD. The substrate profile of Salmonella AcrD differed from that of E. coli AcrD. Mutations targeting the previously defined PAP-RND interaction sites in AcrA/AcrE impaired efflux of AcrD-dependent substrates. Conclusions These data indicate that AcrE forms an efflux-competent pump with AcrD and thus presents an alternative PAP for this pump. Mutagenesis of the conserved RND binding sites validates the interchangeability of AcrA and AcrE, highlighting them as potential drug targets for efflux inhibition.

scholarly journals Functional Cloning and Characterization of a Multidrug Efflux Pump, MexHI-OpmD, from a Pseudomonas aeruginosa Mutant

2003 ◽  
Vol 47 (9) ◽  
pp. 2990-2992 ◽  
Author(s):  
Hiroshi Sekiya ◽  
Takehiko Mima ◽  
Yuji Morita ◽  
Teruo Kuroda ◽  
Tohru Mizushima ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Ethidium Bromide ◽  
Rhodamine 6G ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Multidrug Efflux ◽  
Chromosomal Dna ◽  
Multidrug Efflux Pump ◽  
Multidrug Efflux Pumps

ABSTRACT We isolated mutant YM644, which showed elevated resistance to norfloxacin, ethidium bromide, acriflavine, and rhodamine 6G, from Pseudomonas aeruginosa YM64, a strain that lacks four major multidrug efflux pumps. The genes responsible for the resistance were mexHI-opmD. Elevated ethidium extrusion was observed with cells of YM644 and YM64 harboring a plasmid carrying the genes. Disruption of the genes in the chromosomal DNA of YM644 made the cells sensitive to the drugs.

scholarly journals Molecular Mechanism of MBX2319 Inhibition of Escherichia coli AcrB Multidrug Efflux Pump and Comparison with Other Inhibitors

2014 ◽  
Vol 58 (10) ◽  
pp. 6224-6234 ◽  
Author(s):  
Attilio V. Vargiu ◽  
Paolo Ruggerone ◽  
Timothy J. Opperman ◽  
Son T. Nguyen ◽  
Hiroshi Nikaido
Keyword(s):  
Efflux Pump ◽  
Efflux Pumps ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Efflux Pump Inhibitor ◽  
Content Type ◽  
X Ray Crystallography ◽  
Resistance Nodulation Division ◽  
Hydrophobic Portion ◽  
Dynamics Simulations

ABSTRACTEfflux pumps of the resistance nodulation division (RND) superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. The development of inhibitors of the RND pumps would improve the efficacy of current and next-generation antibiotics. To date, however, only one inhibitor has been cocrystallized with AcrB. Thus,in silicostructure-based analysis is essential for elucidating the interaction between other inhibitors and the efflux pumps. In this work, we used computer docking and molecular dynamics simulations to study the interaction between AcrB and the compound MBX2319, a novel pyranopyridine efflux pump inhibitor with potent activity against RND efflux pumps ofEnterobacteriaceaespecies, as well as other known inhibitors (D13-9001, 1-[1-naphthylmethyl]-piperazine, and phenylalanylarginine-β-naphthylamide) and the binding of doxorubicin to the efflux-defective F610A variant of AcrB. We also analyzed the binding of a substrate, minocycline, for comparison. Our results show that MBX2319 binds very tightly to the lower part of the distal pocket in the B protomer of AcrB, strongly interacting with the phenylalanines lining the hydrophobic trap, where the hydrophobic portion of D13-9001 was found to bind by X-ray crystallography. Additionally, MBX2319 binds to AcrB in a manner that is similar to the way in which doxorubicin binds to the F610A variant of AcrB. In contrast, 1-(1-naphthylmethyl)-piperazine and phenylalanylarginine-β-naphthylamide appear to bind to somewhat different areas of the distal pocket in the B protomer of AcrB than does MBX2319. However, all inhibitors (except D13-9001) appear to distort the structure of the distal pocket, impairing the proper binding of substrates.

scholarly journals CCCP experimental evolution ofEscherichia coliselects for mutations that increase EmrA activity or that downregulate other PMF-driven drug efflux pumps

2018 ◽  
Author(s):  
Jessie M. Griffith ◽  
Preston J. Basting ◽  
Katarina M. Bischof ◽  
Erintrude P. Wrona ◽  
Karina S. Kunka ◽  
...  
Keyword(s):  
Experimental Evolution ◽  
Efflux Pump ◽  
Enteric Bacteria ◽  
Efflux Pumps ◽  
Proton Motive Force ◽  
Motive Force ◽  
Drug Efflux ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
K 12

ABSTRACTExperimental evolution was conducted withEscherichia coliK-12 W3110 in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler of the proton motive force (PMF). Cultures were serially diluted daily 1:100 in broth medium containing 20-150 μM CCCP at pH 6.5 or at pH 8.0. After 1,000 generations, all populations showed 5- to 10-fold increase in CCCP resistance. Sequenced isolates showed mutations inemrABor in its negative repressormprA; the EmrAB-TolC multidrug efflux pump confers resistance to CCCP and nalidixic acid. Deletion ofemrAabolished the CCCP resistance of these strains. One CCCP-evolved isolate lackedemrAormprAmutations; this strain (C-B11-1) showed mutations in drug efflux regulatorscecR(ybiH) (upregulates drug pumps YbhG and YbhFSR) andgadE(upregulates drug pumpmdtEF). AcecR∷kanRdeletion conferred partial resistance to CCCP. A later evolved descendant of the C-B11 population showed mutations inybhR(MDR efflux). Another isolate showedacrB(MDR efflux pump). TheacrBisolate was sensitive to chloramphenicol and tetracycline, which are effluxed by AcrAB. Other mutant genes in CCCP-evolved strains includeadhE(alcohol dehydrogenase),rng(ribonuclease G), andcyaA(adenylate cyclase). Overall, experimental evolution revealed a CCCP fitness advantage for mutations increasing its own efflux via EmrA; and for mutations that may decrease proton-driven pumps that efflux other drugs not present (cecR, gadE, acrB, ybhR). These results are consistent with our previous report of drug sensitivity associated with evolved tolerance to a partial uncoupler (benzoate or salicylate).IMPORTANCEThe genetic responses of bacteria to depletion of proton motive force, and their effects on drug resistance, are poorly understood. Our evolution experiment reveals genetic mechanisms of adaptation to the PMF uncoupler CCCP, including selection for and against various multidrug efflux pumps. The results have implications for our understanding of the gut microbiome, which experiences high levels of organic acids that decrease PMF. Organic acid uncouplers may select against multidrug resistance in evolving populations of enteric bacteria.

scholarly journals Chlorpromazine and Amitriptyline Are Substrates and Inhibitors of the AcrB Multidrug Efflux Pump

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Elizabeth M. Grimsey ◽  
Chiara Fais ◽  
Robert L. Marshall ◽  
Vito Ricci ◽  
Maria Laura Ciusa ◽  
...  
Keyword(s):  
De Novo ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Inhibition Mechanism ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Inhibitory Compounds ◽  
Serovar Typhimurium ◽  
Rnd Efflux Pumps

Efflux pumps of the resistance nodulation-cell division (RND) superfamily are major contributors to multidrug resistance for most of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. The development of inhibitors of these pumps would be highly desirable; however, several issues have thus far hindered all efforts at designing new efflux inhibitory compounds devoid of adverse effects. An alternative route to de novo design relies on the use of marketed drugs, for which side effects on human health have been already assessed. In this work, we provide experimental evidence that the antipsychotic drugs chlorpromazine and amitriptyline are inhibitors of the AcrB transporter, the engine of the major RND efflux pumps in Escherichia coli and Salmonella enterica serovar Typhimurium. Furthermore, in silico calculations have provided a molecular-level picture of the inhibition mechanism, allowing rationalization of experimental data and paving the way for similar studies with other classes of marketed compounds.

scholarly journals Molecular Cloning and Characterization of an ABC Multidrug Efflux Pump, VcaM, in Non-O1 Vibrio cholerae

2003 ◽  
Vol 47 (8) ◽  
pp. 2413-2417 ◽  
Author(s):  
Nazmul Huda ◽  
Eun-Woo Lee ◽  
Jing Chen ◽  
Yuji Morita ◽  
Teruo Kuroda ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Vibrio Cholerae ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Atp Binding ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Hoechst 33342 ◽  
Hydrophobic Domain ◽  
Multidrug Efflux Pumps

ABSTRACT A gene responsible for multidrug resistance was cloned from the chromosomal DNA of non-O1 Vibrio cholerae NCTC 4716 by using as a host drug-hypersensitive Escherichia coli strain KAM32, which lacks major multidrug efflux pumps. E. coli cells transformed with the gene showed elevated levels of resistance to a number of structurally dissimilar drugs, such as tetracycline, norfloxacin, ciprofloxacin, doxorubicin, daunomycin, 4′,6-diamidino-2-phenylindole, and Hoechst 33342. We determined the nucleotide sequence and found one open reading frame. We designated the gene vcaM. The deduced product, VcaM, seems to be a polypeptide with 619 amino acid residues (69 kDa) that has a putative topology of six transmembrane segments in the N-terminal hydrophobic domain, followed by an ATP binding domain in the C-terminal hydrophilic region. The sequence of VcaM was shown to be similar to those of human multidrug resistance proteins P-glycoprotein MDR1 and lactococcal LmrA, which are driven by ATP. The efflux of Hoechst 33342 and doxorubicin from cells possessing VcaM was detected. The efflux activity was inhibited by reserpine and sodium o-vanadate, which are potent inhibitors of MDR1 and LmrA. Thus, we conclude that VcaM is a member of the family of multidrug efflux pumps of the ATP binding cassette type and the first experimentally proven example of a multidrug efflux pump of this family in gram-negative bacteria.

scholarly journals Spermidine and spermine are the natural substrates of the Acinetobacter baumannii AmvA multidrug efflux pump

2020 ◽  
Author(s):  
Francesca L. Short ◽  
Qi Liu ◽  
Heather E. Ashwood ◽  
Varsha Naidu ◽  
Liping Li ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Transcriptional Responses ◽  
Multiple Gene ◽  
Evolutionary Origins ◽  
Multidrug Efflux Pumps ◽  
Long Chain

AbstractMultidrug efflux pumps are important drivers of antibiotic resistance in Acinetobacter baumannii and other pathogens, however their ‘natural’ roles beyond transport of clinical antimicrobials are poorly described. Polyamines are an ancient class of molecules with broad roles in all three kingdoms of life, and are the likely natural substrate of at least one efflux pump family. We have defined the transcriptome of A. baumannii following treatment with high levels of the polyamines putrescine, cadaverine, spermidine and spermine. These molecules influenced expression of multiple gene classes in A. baumannii including those associated with virulence, and the four polyamines induced distinct but overlapping transcriptional responses. Polyamine shock also induced expression of the MFS-family efflux pump gene amvA and its repressor gene amvR. Loss of amvA dramatically reduced tolerance to the long-chain triaamine spermidine, but caused only modest changes in resistance to known AmvA substrates such as acriflavine. We confirmed reduced accumulation of spermidine in amvA-deficient A. baumannii, and showed that its expression is induced by long-chain polyamines through its cognate regulator AmvR. Our findings suggest that the conserved A. baumannii efflux pump AmvA has evolved to export spermidine from the cell, but that its substrate recognition promiscuity also allows activity against clinically-important biocides and antibiotics.ImportanceAMR genes, including multidrug efflux pumps, evolved long before the ubiquitous use of antimicrobials in medicine and infection control. Multidrug efflux pumps often transport metabolites, signals and host-derived molecules in addition to antibiotics or biocides. Understanding the ancestral physiological roles of multidrug efflux pumps could help to inform the development of strategies to subvert their activity. In this study, we investigated the response of Acinetobacter baumannii to polyamines, a widespread, abundant class of amino acid-derived metabolites, which led us to identify long-chain polyamines as natural substrates of the disinfectant efflux pump AmvA. A second clinically-important efflux pump, AdeABC, also contributed to polyamine tolerance. Our results suggest that the disinfectant resistance capability that allows A. baumannii to survive in hospitals may have evolutionary origins in the transport of polyamine metabolites.

scholarly journals The assembled structure of a complete tripartite bacterial multidrug efflux pump

2009 ◽  
Vol 106 (17) ◽  
pp. 7173-7178 ◽  
Author(s):  
Martyn F. Symmons ◽  
Evert Bokma ◽  
Eva Koronakis ◽  
Colin Hughes ◽  
Vassilis Koronakis
Keyword(s):  
Efflux Pump ◽  
Cell Envelope ◽  
Adaptor Protein ◽  
Drug Binding ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Inner Membrane ◽  
Multidrug Efflux Pumps ◽  
Docking Approach

Bacteria likeEscherichia coliandPseudomonas aeruginosaexpel drugs via tripartite multidrug efflux pumps spanning both inner and outer membranes and the intervening periplasm. In these pumps a periplasmic adaptor protein connects a substrate-binding inner membrane transporter to an outer membrane-anchored TolC-type exit duct. High-resolution structures of all 3 components are available, but a pump model has been precluded by the incomplete adaptor structure, because of the apparent disorder of its N and C termini. We reveal that the adaptor termini assemble a β-roll structure forming the final domain adjacent to the inner membrane. The completed structure enabled in vivo cross-linking to map intermolecular contacts between the adaptor AcrA and the transporter AcrB, defining a periplasmic interface between several transporter subdomains and the contiguous β-roll, β-barrel, and lipoyl domains of the adaptor. With short and long cross-links expressed as distance restraints, the flexible linear topology of the adaptor allowed a multidomain docking approach to model the transporter–adaptor complex, revealing that the adaptor docks to a transporter region of comparative stability distinct from those key to the proposed rotatory pump mechanism, putative drug-binding pockets, and the binding site of inhibitory DARPins. Finally, we combined this docking with our previous resolution of the AcrA hairpin–TolC interaction to develop a model of the assembled tripartite complex, satisfying all of the experimentally-derived distance constraints. This AcrA3-AcrB3-TolC3model presents a 610,000-Da, 270-Å-long efflux pump crossing the entire bacterial cell envelope.

scholarly journals Structure of the periplasmic adaptor protein from a major facilitator superfamily (MFS) multidrug efflux pump

FEBS Letters ◽  
2014 ◽  
Vol 588 (17) ◽  
pp. 3147-3153 ◽  
Author(s):  
Philip Hinchliffe ◽  
Nicholas P. Greene ◽  
Neil G. Paterson ◽  
Allister Crow ◽  
Colin Hughes ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Adaptor Protein ◽  
Major Facilitator Superfamily ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Major Facilitator

scholarly journals Function and Inhibitory Mechanisms of Multidrug Efflux Pumps

2021 ◽  
Vol 12 ◽  
Author(s):  
Kunihiko Nishino ◽  
Seiji Yamasaki ◽  
Ryosuke Nakashima ◽  
Martijn Zwama ◽  
Mitsuko Hayashi-Nishino
Keyword(s):  
Efflux Pump ◽  
Efflux Pumps ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Bacterial Cells ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Inhibitory Mechanisms ◽  
Multidrug Efflux Pumps ◽  
Multiple Antibiotics

Multidrug efflux pumps are inner membrane transporters that export multiple antibiotics from the inside to the outside of bacterial cells, contributing to bacterial multidrug resistance (MDR). Postgenomic analysis has demonstrated that numerous multidrug efflux pumps exist in bacteria. Also, the co-crystal structural analysis of multidrug efflux pumps revealed the drug recognition and export mechanisms, and the inhibitory mechanisms of the pumps. A single multidrug efflux pump can export multiple antibiotics; hence, developing efflux pump inhibitors is crucial in overcoming infectious diseases caused by multidrug-resistant bacteria. This review article describes the role of multidrug efflux pumps in MDR, and their physiological functions and inhibitory mechanisms.

The pseudo-atomic structure of an RND-type tripartite multidrug efflux pump

2015 ◽  
Vol 396 (9-10) ◽  
pp. 1073-1082 ◽  
Author(s):  
Dijun Du ◽  
Jarrod Voss ◽  
Zhao Wang ◽  
Wah Chiu ◽  
Ben F. Luisi
Keyword(s):  
Antimicrobial Agents ◽  
Transmembrane Domain ◽  
Efflux Pump ◽  
Cell Envelope ◽  
Toxic Substances ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Transporter Family ◽  
Wide Range ◽  
Rnd Transporter

Abstract Microorganisms encode several classes of transmembrane molecular pumps that can expel a wide range of chemically distinct toxic substances. These machines contribute to the capacity of the organisms to withstand harsh environments, and they help to confer resistance against clinical antimicrobial agents. In Gram-negative bacteria, some of the pumps comprise tripartite assemblies that actively transport drugs and other harmful compounds across the cell envelope. We describe recent structural and functional data that have provided insights into the architecture and transport mechanism of the AcrA-AcrB-TolC pump of Escherichia coli. This multidrug efflux pump is powered by proton electrochemical gradients through the activity of AcrB, a member of the resistance/nodulation/cell division (RND) transporter family. Crystallographic data reveal how the small protein AcrZ binds to AcrB in a concave surface of the transmembrane domain, and we discuss how this interaction may affect the efflux activities of the transporter.

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